Bone Regeneration Using Gene-Activated Matrices.

HHS Public Access Author manuscript Author Manuscript

AAPS J. Author manuscript; available in PMC 2017 September 01. Published in final edited form as: AAPS J. 2017 January ; 19(1): 43–53. doi:10.1208/s12248-016-9982-2.

Bone regeneration using gene-activated matrices Sheetal D’Melloa,#, Keerthi Atluria,#, Sean M. Gearya, Liu Hongc, Satheesh Elangovanb,*, and Aliasger K. Salema,b,* aDivision

of Pharmaceutics and Translational Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA, 52242

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bDepartment

of Periodontics, College of Dentistry, University of Iowa, Iowa City, IA, 52242

cDepartment

of Prosthodontics, College of Dentistry, University of Iowa, Iowa City, IA, 52242

Abstract

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Gene delivery to bone is a potential therapeutic strategy for directed, sustained and regulated protein expression. Tissue engineering strategies for bone regeneration include delivery of proteins, genes (viral and non-viral-mediated delivery) and/or cells to the bone defect site. In addition, biomimetic scaffolds and scaffolds incorporating bone anabolic agents greatly enhance the bone repair process. Regional gene therapy has the potential of enhancing bone defect healing and bone regeneration by delivering osteogenic genes locally to the osseous lesions, thereby reducing systemic toxicity and the need for using supraphysiological dosages of therapeutic proteins. By implanting gene-activated matrices (GAMs), sustained gene expression and continuous osteogenic protein production in situ can be achieved in a way that stimulates osteogenesis and bone repair within osseous defects. Critical parameters substantially affecting the therapeutic efficacy of gene therapy include: the choice of osteogenic transgene(s), selection of non-viral or viral vectors, the wound environment, and the selection of ex vivo and in vivo gene delivery strategies, such as GAMs. It is critical for gene therapy applications that clinically beneficial amounts of proteins are synthesized endogenously within and around the lesion in a sustained manner. It is therefore necessary that reliable and reproducible methods of gene delivery be developed and tested for their efficacy and safety before translating into clinical practice. Practical considerations such as the age, gender and systemic health of patients and the nature of the disease process also need to be taken into account in order to personalize the treatments and progress towards developing a clinically applicable gene therapy for healing bone defects. This review discusses tissue engineering strategies to regenerate bone with specific focus on non-viral gene delivery systems.

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Keywords Gene activated matrix; Transcript activated matrix; Bone healing; Collagen Scaffold; Plasmid DNA and chemically modified RNA

*

Corresponding Authors: Dr. Aliasger K. Salem, 115 S. Grand Avenue, S228 PHAR, University of Iowa College of Pharmacy, Iowa City, IA 52242., [email protected], Telephone: 319 335 8810, Fax: 319 335 9349. Dr. Satheesh Elangovan, 801 Newton Road, S464, Department of Periodontics, University of Iowa College of Dentistry, Iowa City, Iowa -52242, [email protected], Telephone: 319 335 7543, Fax: 319 335 7239. #Joint First Authors

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1. Bone healing mechanism: understanding the biology of bone and bone tissue formation

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Bone is a dynamic, highly vascularized tissue that provides essential mechanical and structural support to the body. Due to its high degree of vascularization, bone has a strong capacity to heal, supply osteoprogenitor cells and mobilize minerals, especially calcium, when required. Bone healing involves a complex set of events encompassing a large number of genetic and molecular triggers, morphogens, signaling molecules and transcriptional regulators that act in concert during specific stages of the healing process. The process of bone healing starts with the formation of a blood clot, followed by an inflammatory phase to remove potential antigens or foreign material from the wound bed.(1) Any impairment in the clotting process due to local or systemic factors or due to the use of anticoagulation drugs may lead to impaired healing. The blood clot is then gradually replaced by granulation tissue with mediators such as vascular endothelial growth factor (VEGF) and angiopoietins playing a crucial role in this phase.(2) The highly vascularized granulation tissue is subsequently replaced by woven bone that then gradually matures.(1) In the reparative phase, growth factors such as transforming growth factor-β (TGF-β), basic fibroblast growth factor (bFGF), platelet-derived growth factor-BB (PDGF-BB), and insulin-like growth factor-I (IGF-I) contribute to the induction of callus formation within the granulation tissue.(3) There are two major mechanisms by which bone formation takes place. One is endochondral ossification in which endothelial cells proliferate and chondroblasts differentiate in the clot area due to lack of oxygen, ultimately resulting in the formation of hyaline cartilage.(4) Chondroblasts after synthesizing the cartilaginous matrix become hypertrophic and produce VEGF and bFGF. New blood vessels are formed in this region which results in the transport of osteoprogenitor and haematopoietic cells, resulting in the replacement of cartilage with bone and bone marrow.(4) The other process is intramembranous ossification in which preosteoblasts differentiate into osteoblasts which then secrete extracellular matrix proteins and deposit calcium to harden the matrix.(5) The final stage of bone healing is the resorption phase where macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL) mediates the activity of macrophages and osteoclasts in the resorption of hard tissue debris.(6) All these phases together contribute to the formation of a complete remodeled bone which is both structurally and functionally similar to the native bone.

2. Tissue engineering strategies to regenerate bone Author Manuscript

The healing capacity of bone in some situations might be limited or insufficient to heal large bone defects. Approaches such as bone grafting are widely used but have several limitations. Bone substitutes based on novel materials continues to be an area of active research, however, as yet, a successful product with predictable regenerative capacity has not been developed for widespread clinical use. Thus, most bone injuries are still not treated in a predictable manner. Each year there are approximately one million cases of skeletal defects that need bone grafting to achieve union. Current treatments are based on autologous bone grafting which is considered the gold standard approach for bone healing and regeneration. AAPS J. Author manuscript; available in PMC 2017 September 01.

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Autografts are bone grafts transferred from a different part of the patient’s own body, which provide excellent osteoconductive, osteoinductive, and osteogenic properties.(7) However, due to second surgical site morbidity, associated pain, and limited supply, this strategy is reserved for only a restricted number of cases.(7) Grafts originating from a different individual of the same (allografts) and different (xenografts) species have several limitations, including the possibility of immune rejection and pathogen transmission from donor to host. (8) The success rate in terms of graft incorporation is lower for allografts or xenografts when compared to autografts.(9) Alternative sources for bone grafts include synthetic alloplasts made from metals, polymers or ceramics.(7) They too possess certain disadvantages such as poor integration with the native tissue at the site of the defect and potential failure due to fatigue or infection caused during implantation. In addition, ceramics are very brittle and cannot be used in locations of high stress or mechanical load.(10)

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Owing to the drawbacks of bone grafts, over the last few decades, identification of key molecules (such as growth factors, cytokines and morphogens) involved in bone development and fracture healing is in progress and has led to the introduction and rapid expansion of biomimetic materials in the field of bone tissue engineering. Tissue engineering has been defined as “an interdisciplinary field of research that applies the principles of engineering and the life sciences towards the development of biological substitutes that restore, maintain, or improve tissue function”.(11) Successful healing of fractures requires integration of engineered tissue with surrounding host tissues and involves combined input from signaling generated by intercellular communications, cell-matrix interactions and growth factors.

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Tissue-engineering strategies for bone regeneration have included recombinant protein therapy to deliver osteogenic cytokines and growth factors, delivery of nucleic acids (DNA, mRNA) encoding growth factors that promotes bone growth, and transplantation of osteogenic cells at sites of bone defects. Osteogenic cells can be utilized per se or can be either transduced with viruses or transfected with non-viral vectors prior to implantation. These approaches may be further modified by combining with biomimetic biomaterial scaffolds in order to boost the therapeutic response and bone repair process. Scaffolds are supportive substrates that provide a three dimensional platform for the cells to successfully transform into bone tissue. Biomaterial scaffold-based delivery systems need to be designed such that they can control and maintain the activity of the incorporated therapeutics and serve to prolong their residence time. Three-dimensional macroporous scaffolds fabricated from biodegradable polymers can act as osteoconductive substrates upon direct implantation in vivo, recruiting progenitor cells to the wound site.

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a. Growth Factor and Cytokine Protein-based Approaches Growth factors act in a concentration, and time, dependent manner to control cell migration, differentiation and/or proliferation by binding to their specific target cell surface receptors and can thus be to repair damaged tissues (Table 1). Osteoinductivity is achieved by the action of bone morphogenetic proteins (BMPs, BMP-2 to BMP-8), TGF-β and other growth factors that stimulate osteogenesis.(12) Pro-angiogenic factors, such as bFGF, VEGF, PDGF-BB, TGF-β and angiopoietins-1 and 2, contribute to osteoconductivity.(13) Multiple

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growth factors, including both osteoinductive and osteoconductive factors have been shown to synergistically promote angiogenesis and enhance bone regeneration.(14) A brief overview of the pathways through which these growth factors induce osteogenesis and angiogenesis is provided in figure 1. One significant drawback of current protein-based therapies is their lack of specificity for osteoblasts or bone-forming cells. Due to their short half-lives and rapid degradation, high doses of proteins (in milligrams) are necessary for direct clinical application despite very small quantities being required for localized osteoinduction. These supra-physiological doses, in addition to the functional heterogeneity of proteins, may result in unwanted complications. For example, high doses of BMP-2 can result in soft tissue swelling, radiculitis and ectopic bone formation.(15) Owing to these unpredictable adverse effects and high cost, the prospect of protein-based therapies is limited.

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b. Cell-Based Approaches

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Cell therapy is an approach based on transplantation of key cells which can synthesize the desired therapeutic growth factors at the implanted site. Isolated autologous cells that are expanded ex vivo can be genetically modified to produce growth factors and can be transplanted into the defect.(16) Cell-based bone tissue engineering includes both somatic and undifferentiated stem cells.(17) In contrast to stem cells, somatic cells have limited potency, lack self-renewal ability and are committed to the production of only a single cell type. Hence, they have very limited use in complex tissue engineering approaches. As a result, a significant portion of current research is concentrated on the use of stem cells. Human mesenchymal stem cells (MSCs) have been primarily isolated from adipose tissue, bone marrow, umbilical cord, and teeth (dental pulp and periodontal ligaments) and other sources. These cells are inherently capable of differentiating into bone tissue cells under appropriate stimuli including appropriate gene transfection. Genetically-modified MSCs can be cultured to produce varying combinations of growth factors such as bFGF and VEGF, BMP-2 and BMP-7, and VEGF and BMP-4.(18) Before implantation at the defect site, gene transfer to these cells can be performed ex vivo using viral vectors and then combined with polymers, such as collagen type I, to create cell/polymer constructs.(19) Various studies have also reported the use of ex vivo expanded autologous bone marrow-derived osteoprogenitor cells grown on macroporous hydroxyapatite scaffolds for implantation at lesion sites. This treatment, devoid of any cellular genetic modifications, was intended for the repair of large bone defects in long bones and demonstrated repair and functional recovery of segmental bone defects.(20)

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Gene therapy is considered an effective means of delivering growth factors in a sustained fashion whilst overcoming the limitations of using high protein doses. Gene delivery is more cost-effective than bolus protein delivery and it can be finely controlled.(21) Furthermore, simultaneous delivery of multiple genes is possible and customization is relatively straightforward. We have previously evaluated the feasibility and efficacy of co-delivering genes encoding FGF-2 and BMP-2 to promote osteogenesis in human adipose-derived mesenchymal stem cells (hADMSC).(22) We showed that when compared to delivery of


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pDNA (FGF-2) or pDNA (BMP-2) alone, co-delivery of both genes resulted in significant enhancement in the secretion of BMP-2 protein from the transfected hADMSC. Posttransfection, a significantly higher expression of osteogenic markers, such as runx-2 and osteocalcin, was observed in hADMSC that were simultaneously transfected with FGF-2 and BMP-2 genes, compared to delivery of single genes.(22) In vitro mineralization data further confirmed the synergistic effects of co-delivering FGF-2 and BMP-2 genes.(22) Altogether, gene therapy is considered to be an effective alternative to protein therapy. The three essential material components of gene therapy include; pDNA encoding the desired therapeutic protein, a vector to facilitate the cellular uptake of the pDNA, and the in situ or ex vivo target tissue or cells that produce the desired protein upon transfection. a. Non-Viral Gene delivery

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Although viral gene therapy has proven to be efficaceous in several animal studies, perceived safety concerns continue to present challenges with respect to conducting human clinical trials and therefore clinical application of viral gene therapy for non-lethal conditions is generally undesirable. In spite of lower transfection efficiencies compared to that of viral vectors, non-viral vectors are safer and can be clinically translated for potential bone regeneration applications. Hence the main focus of this review is on the utilization of non-viral vectors, with a primary focus on polymers as gene delivery vectors.


Ideally, a vector mediating successful gene therapy should possess a number of crucial attributes that allow for therapeutic levels of transgene expression for an adequate duration of time. The transgenes must be delivered to the target cell nuclei in a selective manner that ensures efficient transfection associated with minimal cytotoxicity and safety concerns. In addition, it should ideally be target cell-specific and have a controllable timeframe of protein expression.(23) Unfortunately, despite their strong safety profiles, non-viral gene delivery systems suffer from having lower transfection efficiencies, which has previously hampered their potential.(24) Tremendous growth in the field of nanotechnology in the last decade has led to the production of safer non-viral vectors with improved transfection efficiencies. Several studies have explored the in vivo and ex vivo delivery of genes (encoding growth factors or transcriptional factors) using non-viral vectors for bone regeneration. Non-viral gene delivery agents have advantages over viral vectors in that the responses to the treatment are less immunogenic, less toxic, and there are lower pathogenic, carcinogenic or other mutagenic concerns, often associated with viral vectors, thus making them safer for clinical applications.(24) Moreover, the gene expression induced is transient and the expression levels can be tightly regulated as needed depending on the acute or chronic nature of the disease. It is also relatively easy to alter and optimize the properties of these gene carriers so as to create a balance between transfection efficiency and cytotoxicity. pDNA can be complexed via electrostatic interactions with liposomes, polymers or other polycations to form either lipoplexes or polyplexes. Among the numerous non-viral gene vectors studied in vitro and in vivo, PEI, especially the branched 25 kDa PEI polymer is one of the most successful gene transfer agents to date.(25) PEI is believed to exhibit higher transfection efficiencies than many other non-viral vectors due to a phenomenon known as the ‘proton sponge effect’(26) and the level of transfection efficiency attained with PEI is considered comparable with that of viral vectors.


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b. In Vivo versus Ex Vivo gene therapy

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There are two main methods of gene transfer for tissue regeneration: 1) transfection of MSCs ex vivo and subsequent transplantation into the defect site,(27) and 2) direct application of osteogenic genes to the defect site in vivo. Plasmids containing cDNA equivalents of therapeutic genes can be delivered per se or the plasmid can be complexed with polycations to form nanoplexes. These methods could be used in conjunction with scaffolds to enhance bone regeneration.

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A high transfection efficiency of host cells is required for the in vivo gene delivery approach to be effective. With this approach, it is difficult to obtain targeted gene delivery to specific cells, as the cells surrounding the target tissue of interest may also be transfected. In ex vivo gene transfer, the isolated and culture-expanded cells can be transfected in vitro and are then implanted into the defect. Ex vivo gene therapy can target specific cell populations of interest and permits selection, control, and scrutiny of the genetically-altered cells prior to re-implantation. However, compared with in vivo gene therapy, ex vivo approaches are generally more surgically invasive, technically complex and expensive, thus reducing their scope for clinical translation.(28) Therefore, there is growing interest in the development of non-viral vectors with higher transfection efficiencies that can deliver single or multiple gene-encoding pDNA in vivo.

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Gene-activated matrices (GAMs) are inert scaffold systems containing viral or non-viral gene delivery vectors that have been widely investigated and used in wound healing and tissue engineering approaches.(29) Following gene delivery, the recombinant protein could be expressed in situ by endogenous wound repair cells. Although secreted in small amounts, protein expression for prolonged periods of time can promote bone regeneration.(30) GAMs temporarily serve as three-dimensional templates for tissue formation and guide the growth of new functional tissue. They can encapsulate and retain the gene within the sponge matrices for longer durations thereby enhancing matrix deposition and blood vessel formation in the developing bone tissue.(31) In contrast, direct injection of the gene mediates shorter cellular expression and does not significantly affect tissue formation.(32) Research performed by our group demonstrated the ability of a GAM comprising a collagen scaffold injected with PEI/pDNA (encoding PDGF-B) complexes to regenerate bone.(33) The in vivo regenerative capacity of the GAM was evaluated in five mm diameter criticalsized calvarial defects in rats and demonstrated a forty four-fold higher new bone volume percentage in calvarial defects when compared to implanted empty scaffolds.(34)

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a. Scaffold design criteria for gene delivery systems GAMs are ideally composed of biodegradable and biocompatible polymeric materials that are bioresorbable upon in vivo implantation into osseous defects. The scaffold matrix provides structural support for osteogenesis or bone remodeling by the infiltrating reparative cells expressing the transgene (Figure 2). Three-dimensional GAMs are typically highly porous in nature and can be fabricated with different desired geometries that can regenerate functional bone tissue while maintaining its original size and shape. A biologically active


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scaffold specifically engineered for bone tissue regeneration must possess a number of key qualities necessary for enabling new tissue formation.(35) Ideally, biomaterials composing GAMs need to be osteoconductive, mechanically compatible and capable of integrating with the surrounding native bone during the repair process. The matrix must be suitable for creating and maintaining appropriate space and a favorable environment in vivo to support tissue development and control the size and shape of the space-filling regenerating tissue. It must also be structurally and mechanically stable, and homogenously porous with optimal pore size suitable for promoting cell adhesion and growth. For bone healing involving guided bone regeneration, it is critical that the scaffold matrices possess suitable physical and mechanical properties for supporting cell survival and proliferation. The ability of the scaffolds to engineer the bone tissue and control its structure is defined by the mechanical properties and degradation rate of the scaffolds. For this reason, the scaffold must maintain architectural integrity after placement in vivo, that is essential for the gradual filling-in of critical-sized defects during the process of bone formation.(36) The biocompatible scaffold must degrade (generating non-toxic degradation products) at a rate such that the new bone tissue is formed, gradually replacing the scaffold. The scaffold should allow for migration and attachment of progenitor cells from the surrounding tissue into the scaffold. It is desirable that the chemical composition of the scaffold contains binding sites (ligands) appropriate for specific cell populations. In addition, the pore size must be large enough to allow maximal cell entry and migration, yet small enough to retain a high specific surface area for cell attachment and matrix deposition.(37) It is also necessary that the scaffolds should possess optimal pore volume fraction (high porosity, usually > 90 %) together with an interconnected pore network for efficient transport of metabolites and nutrients.(38) The pore size, pore shape, and the porosity of the scaffold are critical parameters governing the availability of total specific surface area as well as ligand distribution to the cells. Initiation of the bone regenerative process involves an interplay between the scaffolds and infiltrating cells involving structural, mechanical, and biological cues. For bone regeneration applications, a series of scaffolds with tailored structural, mechanical and biological properties can be fabricated that facilitate cellular adhesion, migration, and tissue development (Table 2). This may significantly enable engineering of the bone tissue with pre-defined structures. b. Soft tissue healing

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Some bone defects, such as those resulting from trauma, also require reconstructive therapy of soft tissue injuries associated with the trauma. This includes repair of blood vessels, cartilage, muscles, ligaments and tendons, neural tissue, and skin. The process of bone formation alone requires the integration of a number of physiological processes such as angiogenesis. New blood vessels are essential for supplying oxygen and providing nutrients necessary to sustain cell vitality required of the highly metabolically active cells involved in repair. They also serve to carry inflammatory and mesenchymal tissue progenitor cells to, and waste and breakdown products from, the wound site.(39) By modifying vascularization through exogenous delivery of various growth factors, including VEGF, PDGF, BMPs and FGF, bone regeneration and healing can be enhanced.(40) This could also be a viable therapeutic approach for healing of soft tissues. The highly vascularized soft tissue envelopes, periosteum and endosteum, restore normal blood supply to the fracture site. By


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combining osteogenic and angiogenic inductive growth factors, complete wound healing can be potentially achieved. Work by Shea et al. showed that polymeric PLGA scaffolds incorporating DNA encoding VEGF resulted in local and sustained delivery of the growth factor at the site of implantation.(41) The treatment led to increased blood vessel density at the local tissue site. In a separate animal study, Mooney at al showed that when pDNA encoding human recombinant PDGF-B was encapsulated into PLGA matrices, an increase in granulation tissue and vascularization was observed.(32) There was a statistically significant increase in the granulation tissue thickness, and the number and area of blood vessels from two to four weeks, thus demonstrating continuous expression of the delivered gene and its promotion of tissue formation over time. Enhancement of skeletal muscle repair was reported by Pierce’s group using collagen-gelatin matrix-immobilized gene vectors encoding either FGF-2 or FGF-6.(42) When delivered to excisional muscle defects, these biomatrices were responsible for promoting angiogenesis that later remodeled to form arteries. Along with enhancing the density of endothelial cells and muscular arterioles at the treatment sites, myotube regeneration and muscle repair was also facilitated. For cartilage engineering applications, Zhang and colleagues carried out a study employing porous chitosan-gelatin scaffolds containing DNA encoding TGF-β1.(43) This GAM proved effective for the proliferation of chondrocytes and also increased the synthesis of major ECM components, thereby promoting cartilage regeneration. The GAM approach can be similarly utilized for gene delivery in the regeneration of other soft tissues.(44) c. Hard tissue regeneration with a focus on the use of GAMs


The engineering of bone tissue requires transient gene expression of growth factors for a time period spanning weeks. A variety of natural, synthetic, and semi-synthetic polymers can be used as substrates for gene delivery. An advantage of employing synthetic polymers as depot systems is that they can be tailored specifically with properties favorable for bone tissue regeneration. These modifications provide control over the amount of pDNA available to cells over time, thus, the time frame of gene expression can be modulated. GAMs may also contain therapeutic gene(s) encapsulated or entrapped into polymeric nanospheres or microspheres for further optimization of release and uptake kinetics. When formulating pDNA into these spherical particles, either the uncondensed (naked) or condensed form (with polycations) can be incorporated.(45) Alternatively, lyophilized pDNA can either be mixed with the polymer particles or pre-encapsulated into polymer microspheres before processing the polymer particles or microspheres into porous scaffolds.(46) The latter approach may result in a more even distribution of pDNA throughout the matrix, with release being regulated by the degrading microspheres. These two approaches may be combined to provide delivery of multiple genes (encoding different growth factors), each with a distinct release rate and delivery kinetics from the same structural scaffold unit. The different phases of bone healing rely on the action of multiple growth factors at distinct stages, for recruitment, proliferation and differentiation of MSCs.(47) Using the aforementioned combinatorial approach for tissue-specific controlled dose and rate of delivery, these signals can be then temporally and spatially manipulated so as to enhance the cellular events necessary for bone regeneration. The formulation properties such as polymer concentration, polymer molecular weight, and the method of preparation can be used to control the loading efficiency, release kinetics, and the bioavailability of the released pDNA.


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The kinetics of gene construct release can also be varied by altering the polymer degradation rate using various polymer formulations. The polymer constructs can be fabricated from synthetic polyesters such as PLGA, PGA, and polycaprolactone, and natural polymers such as chitosan, alginate, hydroxyapatite, calcium phosphate, collagen, and hyaluronan (Table 3). These polymers can also be used in combination.(48)

5. Gene-activated titanium surfaces for enhanced osseointegration

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In dentistry, the introduction of dental implants has revolutionized the way oral rehabilitation and reconstruction is achieved. Through osseointegration, the process by which the titanium surface of the dental implants forms direct bonding with the surrounding bone, dental implants are able to support and retain dental prostheses improving patient’s esthetics and function in a significant manner.(49, 50) Though osseointegration is a highly predictable process, failure does occur and ways to enhance osseointegration are constantly being explored.(51) Our group developed a novel approach of coating the titanium surface with polymer-pDNA nanoplexes to enhance osseointegration.(52) We showed that discs coated with PEI-pDNA (BMP-2) nanoplexes prepared at an N/P ratio of 10 (N/P-10) resulted in 75% cell viability and 14% transfection efficiency in bone marrow stromal cells (BMSCs) in vitro. Compared to controls, the transfection of BMSCs by PEI-pDNA (BMP-2) nanoplexes from the titanium surface resulted in enhanced expression of osteogenic markers including, runx-2, alkaline phosphatase and osteocalcin.(52) Compared to controls, enhanced calcium deposition as determined by qualitative (alizarin red staining) and quantitative (atomic absorption spectroscopy) assays was noted in transfected cells on day 30 post-treatment.(52) The results highlight the potential of this novel approach of using gene-activated titanium surfaces to enhance osseointegration.

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6. RNA-activated matrices for bone regeneration

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As discussed earlier in this review, the inherent barrier to non-viral gene delivery is the need for the polymer-pDNA complex or the pDNA alone to cross the nuclear membrane to enter the nucleus, where they utilize the machinery for transcription of the encoded DNA into mRNA. This rate limiting step which requires the target cells to be in a constantly dividing state to allow for nuclear entry significantly affects the overall transfection efficiency of the non-viral gene delivery system and also minimizes its applications. We have shown that synthesizing and utilizing chemically modified RNA (cmRNA) encoding BMP-2 instead of pDNA as the active molecule in combination with PEI as a vector resulted in significantly higher bone regeneration in a rat calvarial defect model.(53) Issues related to stability and immunogenicity, inherent to RNA were addressed by strategic modifications of RNA. The PEI-cmRNA (BMP-2) outperformed PEI-pDNA (BMP-2) across a wide range of in vitro and in vivo based metrics.(53) This study and a subsequent investigation that validated these findings, may pave the way for a new field of chemically modified RNA based therapy for bone regeneration.(54)


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7. Advantages and disadvantages of GAMs for bone regeneration: Biocompatibility/safety of GAMs


Utilizing non-viral GAMs for tissue engineering has been shown to be effective in generating sustained expression of recombinant growth factors by transfected infiltrating wound repair cells, as well as being more cost-effective, and may be safer for use clinically when compared to recombinant protein or viral therapy. The implanted GAMs facilitate gene expression and protein production for an extended period of time thereby stimulating an enhanced therapeutic response for osteogenesis and bone repair. Localized gene therapy has also been reported to eliminate or reduce systemic toxicity resulting from dose dumping which can occur with current protein therapies.(55, 56) The in vitro production of pDNA is relatively simple, produced in large quantities and economical as compared to commercial protein production.(57) In addition, it has been shown that localized gene delivery using the GAM approach directs the production of endogenous proteins which are less altered and thereby less immunogenic, in a targeted, controlled manner at the site of implantation. The in situ production of proteins by transfected bone repair cells ensures efficient cell surface receptor targeting. Consequently, significantly lower doses of proteins are required to attain similar or even higher levels of therapeutic effect for enhanced bone regeneration, when compared to protein delivery. With GAMs, a long term healing effect due to relatively sustained transgene expression can be achieved in vivo.(58) Gene transfer from GAMs causes specific cells to be differentiated into desired, selected cell types over time and eliminates the need for repeated administrations.(59) Furthermore, incorporation of gene vectors into the porous scaffold affords protection from physiological degradation and delays clearance from the wound site thereby maintaining long term availability of transgenes to cells infiltrating the GAMs. However, there is a lack of knowledge with respect to the amount of proteins actually produced by transfected cells and the amount needed for therapeutic effects in a given clinical situation. Further research is required to determine optimal levels and timing of protein expression needed during the bone healing process. Regarding clinical applications, the lack of preclinical data in large animal models due to the highly expensive and time consuming nature of the studies continues to be a major barrier. Even if encouraging data is obtained readily by simple, available technologies, the funding and the regulatory environment raise issues and pose additional impediments to progress. As mentioned earlier, compared to viral gene delivery systems, GAM based non-viral gene delivery systems have the best potential to overcome key barriers to clinical translation, so long as safety and efficacy can be demonstrated using these materials in appropriate animal and human studies in the future.

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8. Future prospects and challenges in clinical applications Critical-sized bone defects may not be capable of self-healing and regeneration without intervention. This necessitates treatments that facilitate rapid new bone development and reduce healing times. Gene therapy is being investigated in pre-clinical models as a way to treat tissue loss and enhance the regenerative process. Gene transfer can be performed using a variety of viral and non-viral vectors as gene carriers. Depending on the type of defect, either in vivo or ex vivo methods can be implemented. In terms of safety, and therefore


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potential for clinical translation, non-viral vectors may be preferred over viral vectors. In bone tissue engineering, GAM technology is a highly versatile approach for affecting sustained localized gene delivery and extended expression of tissue inductive growth factors. The polymeric scaffold design of GAMs can control tissue development by controlling the release and maintaining sufficient availability of growth factors to the site of injury. The GAM design provides a powerful and useful tool to study, regulate and manipulate cell functions in the bone developmental process thereby providing important details for our understanding of the biology of bone tissue formation and regeneration.

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Although the combination of tissue engineering scaffolds with gene therapy has immense potential to target many cellular processes involved in promoting bone regeneration there are several challenges which need to be addressed. One challenge is to fabricate matrices that possess all the desired properties needed to achieve optimal bone regeneration such as porosity, biocompatibility, mechanical integrity, mechanical stability, osteoconductivity and osteoinductivity. Novel matrices need to be developed that are capable of maintaining structural integrity whilst simultaneously providing ease of access to infiltrating bone progenitors. When compared to the viral vectors, polymeric vectors generally display inferior transfection efficiencies. Hence there is a need to improve upon current formulations, possibly through mimicking viral modes of transduction, such that GAMs possess the ability to transfect cells at levels comparable to viral vectors.

Acknowledgments Our research has been supported by an NIH NIDCR grant (1R21DE024206-01A1), an Osteology Foundation Grant (12-054), and the Lyle and Sharon Bighley Professorship.

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Author Manuscript Figure 1.


Schematic demonstrating the signaling networks and their cross talks involved in differentiating the mesenchymal stem cells (MSCs) into osteoblasts. TGF-β induces osteogenic differentiation by activating PI3K, SMAD 2/3 and RhoA pathways.(100) FGF-2 and BMP-2 can synergistically transform mesenchymal stem cells to osteoblasts via RAS/RAF/MEK/MAPK pathway and a SMAD pathway.(101) PDGF triggers angiogenesis through cross talk between MAPK, Rho/Rac, STAT3 and PI3K pathways.(102) IGF can induce osteogenesis by activating MAPK and PI3K pathways.(103) VEGF by activating PLC, IP3 and FAK pathways can induce angiogenesis.(104, 105) WNT signaling through βcatenin can induce osteogenic differentiation of MSCs.(106) Here, PI3K – Phosphatidylinositol-4,5-bisphosphate 3-kinase, Akt/PKB – Protein kinase B, SMAD – Suppressor of mothers against decapentaplegic, RhoA – Ras homolog gene family, member A, RUNX2 - Runt-related transcription factor 2, MAPK – Mitogen-activated protein kinase, STAT – Signal transducer and activator of transcription, PLC – Phosphoinositide phospholipase C, DAG – Diacylglycerol, IP3 – Inositol 1,4,5-trisphosphate, NOS – Nitric oxide synthase and FAK – Focal adhesion kinase.

Author Manuscript AAPS J. Author manuscript; available in PMC 2017 September 01.

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Figure 2.

Schematic of the gene activated matrices (GAMs) demonstrating the proposed mechanism of action for bone regeneration.


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Table I

Author Manuscript

A list of growth factors and their role in bone regeneration. Growth factor/Protein

Known functions

EGF(60)

Growth and proliferation of mesenchymal and fibroblast cells, induction of granulation tissue formation and angiogenesis

PDGF-BB(61)

Chemo-attractant, induces mitogenesis, promotes angiogenesis, and extracellular matrix synthesis and deposition

TGF-β(62)

Regulation of cell proliferation, differentiation, and matrix synthesis

rhBMP(63)

Stimulation of angiogenesis and migration, induces proliferation and differentiation of mesenchymal stem cells into cartilage and bone-forming cells

VEGF(64)

Promotion of chemotaxis of MSCs, indirectly induces proliferation and differentiation of osteoblast precursor cells

bFGF(65, 66)

Stimulation of migration and proliferation of endothelial cells, hypertrophic chondrocyte differentiation, and osteoblast/osteoclast recruitment to the growth plate

Activin(67)

Induction of osteoblastic cell proliferation and collagen synthesis

IGF-I(68)

Induction of cellular proliferation and matrix synthesis

Author Manuscript

EGF: Epidermal Growth Factor PDGF-BB: Platelet Derived Growth Factor-BB TGF-β: Transforming Growth Factor Beta rhBMP: Recombinant Human Bone Morphogenetic Proteins VEGF: Vascular Endothelial Growth Factor bFGF: Basic Fibroblast Growth Factor IGF-1: Insulin-like Growth Factor 1


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Table II

Author Manuscript

A list of different polymeric scaffolds implicated in tissue engineering applications with their respective modifications.

Author Manuscript Author Manuscript Author Manuscript

Scaffold material

Modification description

Outcome

Collagen-GAG(69, 70)

Addition of GAG to collagen scaffolds, constant cooling rate during the freezing process prior to lyophilization

GAG effectively improves attachment, migration, and infiltration of cells throughout the porous scaffold; uniform porous structure and less variation in mean pore size

Collagen-GAG(71)

Collagen concentration (0.25 %, 0.5 %, and 1 %) and cross-link density (dehydrothermal crosslinking processes at 105 °C for 24 h and 150 °C for 48 h)

Significant improvement in the biological and mechanical properties of the scaffold with increased collagen amount (1 %) and crosslinking (at 150 °C for 48 h); enhanced pore size, permeability, compressive strength, cell number, and cell metabolic activity

Hyaluronic acid-based polymer(72–74)

Chemical modification through total esterification of carboxyl groups

Insoluble polymer with good stability against acidic hydrolysis; covalent binding of hydroxyl functional moieties; promotes cell adhesion, proliferation, ECM production, osteogenic differentiation, and mineralization

Hydroxyapatite/β-tricalcium phosphate ceramic implants(75)

MSCs loaded onto the porous carrier

Stronger bone formation superior to the carrier alone

Collagen-PGA(76, 77)

Collagen sponge mechanically reinforced by incorporation of PGA fiber (dehydrothermal cross-linking)

Enhancement in compression strength; sustained release of pDNA complex; significant attachment of fibroblasts, greater cell proliferation and infiltration; reduction in sponge shrinkage

Gelatin-PLGA(78)

PLGA microspheres loaded into gelatin scaffolds

Increased mechanical strength and flexibility; delivery of multiple genes with distinct release kinetics

PLA, PGA, PLGA(79)

Type of polymer, molecular weight, intrinsic viscosity

High porosity with low molecular weight, PLGA with low intrinsic viscosity; superior mechanical properties with higher lactic acid content

PLGA(80)

Partial fusion of NaCl porogen in the solvent casting-particulate leaching process

Scaffolds with enhanced pore interconnectivity and compressive modulus

PLLA(81)

Scaffold surface modification using gelatin spheres as porogen

Higher compressive modulus; significant improvement in initial cell adhesion and proliferation, cell spreading and matrix secretion

Hyaluronan(82)

Modification with gelatin using disulfide crosslinking

Hyaluronan-gelatin sponge promoted cell attachment, growth, and spreading

PLGA(83)

Coating PLGA microspheres with polydopamine

Increased incorporation and slowed release of pDNA from the scaffold

Collagen(84)

Calcium-phosphate coating for collagen scaffolds

Improved mechanical properties (higher compressive modulus/stiffness)

Collagen(85, 86)

Nano-hydroxyapatite inclusion in the scaffold

Enhanced cell function and osteointegration; significantly increased scaffold stiffness and pore interconnectivity

PCL(87)

Coupling resveratrol through a hydrolysable covalent bond with the carboxylic acid groups on PCL surface grafted with acrylic acid

Significant increase in osteogenesis

Alginate(88)

Mixing octacalcium phosphate (OCP) with alginate solution

Increased elastic modulus and pore size with increasing OCP concentration

Collagen(58)

Specific binding of biotinylated PEI-pDNA complexes to avidin-modified collagen

Enhanced transfection efficiency by immobilizing complexes in the matrix through biotin/avidin bond; inhibits aggregation of


D’Mello et al.

Scaffold material

Page 22

Modification description

Outcome

Author Manuscript

complexes; higher loading efficiency and bioavailability of complexes

Author Manuscript Author Manuscript Author Manuscript AAPS J. Author manuscript; available in PMC 2017 September 01.

D’Mello et al.

Page 23

Table III

Author Manuscript

A list of different types of GAMs investigated for induction of bone formation

Author Manuscript

Scaffold material

Vector

Transgene

Model

Collagen(89)

pDNA

PTH 1–34 or/and BMP-4

Rat femoral defect

Collagen(29)

pDNA

PTH 1–34

Dog tibial defect

PLGA(90)

PEI-pDNA complexes

BMP-4

Rat cranial defect

Poly(propylene fumarate)(91)

Triacrylate/amine polycationic polymer-pDNA polyplexes complexed with gelatin microparticles

BMP-2

Rat cranial defect

Collagen(34)

PEI-pDNA complexes

PDGF-B

Rat cranial defect

Collagen or autologous bone graft(92)

BMP-2 condensed with liposomal vector

BMP-2

Pig cranial defect

Collagen(93)

CaP-pDNA precipitates

BMP-2

Rat tibial defect

Collagen(94)

pDNA

PTH 1–34

Lumbar interbody fusions in sheep

Collagen(95)

pDNA

VEGF165

Rabbit radial defect

Calcium phosphate cement(96)

pDNA complexed with poly(ethyleneglycol) (PEG)-blockpolycation

caALK6 and Runx2

Mouse cranial defect

Collagen(97)

pDNA

Osteogenic protein-1 (OP-1 or BMP-7)

Rat lumbar interbody arthrodesis

Collagen/calciumphosphate(98)

pDNA complexed with PAMAM dendrimer

VEGF165

Mouse intra-femoral defect

Hydroxyapatite(99)

pDNA condensed with cationic liposomes

BMP-2

Rabbit cranial defect

(PEG-b-P[Asp-(DET)]): PEG-b-polyasparagine carrying the N-(2-aminoethyl) aminoethyl group (CH2)2NH(CH2)2NH2 as the side chain) caALK6: constitutively active form of activin receptor-like kinase 6

Author Manuscript

Runx2: runt-related transcription factor 2

Author Manuscript AAPS J. Author manuscript; available in PMC 2017 September 01.

Engineered decellularized matrices to instruct bone regeneration processes.

Chemically modified RNA activated matrices enhance bone regeneration.

Bone regeneration using the principle of guided tissue regeneration.

Bone Regeneration Using Bone Morphogenetic Proteins and Various Biomaterial Carriers.

Oxygen Tension-Controlled Matrices with Osteogenic and Vasculogenic Cells for Vascularized Bone Regeneration In Vivo.

Gene-activated matrices for bone and cartilage regeneration in arthritis (GAMBA).

Bioresorbable zinc hydroxyapatite guided bone regeneration membrane for bone regeneration.

Vertical bone regeneration using rhBMP-2 and VEGF.

QUANTITATIVE EVALUATION OF EXPERIMENTAL BONE REGENERATION USING INDENTATION TESTS.

Recent advances in bone regeneration using adult stem cells.

Cellular bone matrices: viable stem cell-containing bone graft substitutes.

Bone regeneration around a failing implant using guided tissue regeneration. A case report.

Nanomaterials and bone regeneration.

MicroRNAs and Bone Regeneration.

Mandibular bone regeneration after bone slat technique.

Guided bone regeneration using autogenous tooth bone graft in implant therapy: case series.

Salicylic Acid-Based Polymers for Guided Bone Regeneration Using Bone Morphogenetic Protein-2.

Delivery of bone morphogenetic protein-2 and substance P using graphene oxide for bone regeneration.

Stem Cells in Bone Regeneration.

Biophysical Stimulation for Bone Regeneration.

Vascularization strategies for bone regeneration.

Bone tissue engineering and regeneration.

Scaffold design for bone regeneration.

Malware analysis using visualized image matrices.